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Cassava brown streak disease (CBSD) has occurred in the Indian Ocean coastal lowlands and some areas of Malawi in East Africa for decades, and makes the storage roots of cassava unsuitable for consumption. CBSD is associated with Cassava brown streak virus (CBSV) and the recently described Ugandan cassava brown streak virus (UCBSV) [picorna-like (+)ssRNA viruses; genus Ipomovirus; family Potyviridae]. This study reports the first comprehensive analysis on how evolution is shaping the populations of CBSV and UCBSV. The complete genomes of CBSV and UCBSV (four and eight isolates, respectively) were 69.0-70.3 and 73.6-74.4% identical at the nucleotide and polyprotein amino acid sequence levels, respectively. They contained predictable sites of homologous recombination, mostly in the 3'-proximal part (NIb-HAM1h-CP-3'-UTR) of the genome, but no evidence of recombination between the two viruses was found. The CP-encoding sequences of 22 and 45 isolates of CBSV and UCBSV analysed, respectively, were mainly under purifying selection; however, several sites in the central part of CBSV CP were subjected to positive selection. HAM1h (putative nucleoside triphosphate pyrophosphatase) was the least similar protein between CBSV and UCBSV (aa identity approx. 55%). Both termini of HAM1h contained sites under positive selection in UCBSV. The data imply an on-going but somewhat different evolution of CBSV and UCBSV, which is congruent with the recent widespread outbreak of UCBSV in cassava crops in the highland areas (>1000 m above sea level) of East Africa where CBSD has not caused significant problems in the past.

Isolates of two distinct begomovirus species, the severe strain of the species Tomato leaf curl New Delhi virus (tomato leaf curl New Delhi virus-[India:New Delhi:Severe:1992]; ToLCNDV-[IN:ND:Svr:92], bipartite) and the Varanasi strain of the species Tomato leaf curl Gujarat virus (tomato leaf curl Gujarat virus-[India:Varanasi:2001]; ToLCGV-[IN:Var:01], mono/bipartite) infect tomato (Lycopersicon esculentum) and cause severe yield losses in northern India. This study investigated the infectivity properties of genomic components of these two species. Both pseudorecombinants were infectious in Nicotiana benthamiana, Nicotiana tabacum and L. esculentum. Enhanced pathogenicity was observed when DNA-A of ToLCNDV-[IN:ND:Svr:92] was trans-complemented with ToLCGV-[IN:Var:01] DNA-B, and was consistently associated with an increase in accumulation of ToLCGV-[IN:Var:01] DNA-B. Mixed infection of ToLCNDV-[IN:ND:Svr:92] and ToLCGV-[IN:Var:01] always showed extremely severe symptoms, suggesting a synergistic interaction between these two viruses. Southern blot analysis of viral DNAs from infected plants showed a significantly higher level of accumulation of both ToLCNDV components and DNA-B of ToLCGV-[IN:Var:01] with no alteration to levels of DNA-A of ToLCGV-[IN:Var:01]. Symptom development and/or higher infectivity of the supervirulent pseudorecombinants correlated with the increased levels of DNA-B accumulation. Protoplast replication assays revealed that enhanced infectivity by the pseudorecombinant occurred at the level of replication, as DNA-A of ToLCNDV-[IN:ND:Svr:92] enhanced ToLCGV-[IN:Var:01] DNA-B replication, whose accumulation was in turn increased by ToLCGV-[IN:Var:01] DNA-A. This is the first report demonstrating a virulent pseudorecombinant between two distinct species of begomoviruses that infect tomato, and is the second report on synergism between begomoviruses. The results revealed that ToLCGV-[IN:Var:01] DNA-B is capable of associating with different DNA-A components, despite having different iteron sequences.

The full-length genome of a begomovirus and its cognate DNA-beta satellite component associated with chilli leaf curl disease (ChLCD), originating from Varanasi, India, were cloned. Sequence analysis revealed that the viral genome (EF190217) is 2,750 bp and the DNA-beta satellite (EF190215) is 1,361 bp in length. Agroinoculation with partial tandem repeats of the viral genome along with the satellite induced symptoms typical of ChLCD in chilli and Nicotiana benthamiana. However, symptom expression was delayed and milder when the viral genome was agroinoculated alone in these hosts. Sequence comparisons revealed that the genome had the highest sequence identity (95%) with that of chilli leaf curl virus-PK[PK:Mul:98]. The DNA-beta satellite shared maximum sequence identity (88%) with a DNA-beta satellite associated with tomato leaf curl disease from Rajasthan (ToLCBDB-[IN:Raj:03]). These results demonstrate that ChLCD is caused by a complex consisting of the monopartite chilli leaf curl virus and a DNA-beta satellite component. This is the first experimental demonstration of Koch's postulates using cloned DNA molecules associated with chilli leaf curl disease.

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.

Genomes of two isolates of tomato leaf curl geminivirus from India (ToLCV-India) have been sequenced. ToLCV-India contains A and B components, both of which are required for systemic movement and symptom development. The two isolates have 94% sequence identify but one isolate gave mild symptoms in Nicotiana benthamiana and tomato. The genome organization of ToLCV-India is similar to other whitefly-transmitted geminiviruses (WTGs) with bipartite genomes. However, it contains an additional ORF, AV3, that has not been reported for other WTGs. Its coat protein (CP) sequence is highly homologous to that of Indian cassava mosaic virus (90%). Two mutations that truncated the CP after amino acids 65 or 172 did not affect systemic movement and symptom development in either N. benthamiana or tomato. However, the symptoms caused by mutant viruses were different from those in plants infected with unmodified viruses, and plants infected with the mutants had markedly reduced amounts of single-stranded viral DNA. Comparison of sequences and other biological features of ToLCV-India with other geminiviruses showed that ToLCV-India is a distinct virus and is related to the WTGs from the Old World. It is similar to African cassava mosaic virus in its requirement for B component and dispensability of coat protein for symptom development, unlike other geminiviruses that infect tomato in the Old World. It is proposed that ToLCV-India evolved more recently as compared to other geminiviruses that infect tomato in the Old World.